microbiology reviews

38 Pages·2014·1.86 MB·English

by  Robert Linhardt

REVIEW ARTICLE
Masquerading microbial pathogens: capsular polysaccharides
mimic host-tissue molecules
Brady F. Cress1, Jacob A. Englaender2, Wenqin He1, Dennis Kasper3, Robert J. Linhardt1,2,4 &
Mattheos A.G. Koffas1,2
1DepartmentofChemicalandBiologicalEngineering,CenterforBiotechnologyandInterdisciplinaryStudies,RensselaerPolytechnicInstitute,
Troy,NY,USA;2DepartmentofBiology,CenterforBiotechnologyandInterdisciplinaryStudies,RensselaerPolytechnicInstitute,Troy,NY,USA;
3DepartmentofMicrobiologyandImmunobiology,HarvardMedicalSchool,Boston,MA,USA;and4DepartmentofChemistry,ChemicalBiology,
CenterforBiotechnologyandInterdisciplinaryStudies,RensselaerPolytechnicInstitute,Troy,NY,USA
Correspondence:MattheosKoffasand Abstract
RobertLinhardt,RensselaerPolytechnic
The increasing prevalence of antibiotic-resistant bacteria portends an impend-
Institute,Troy,NY,USA.
e-mail:[email protected] ing postantibiotic age, characterized by diminishing efficacy of common antibi-
[email protected] otics and routine application of multifaceted, complementary therapeutic
approaches to treat bacterial infections, particularly multidrug-resistant organ-
Received25June2013;revised16October isms. The first line of defense for most bacterial pathogens consists of a physi-
2013;accepted19December2013.Final
cal and immunologic barrier known as the capsule, commonly composed of a
versionpublishedonline27January2014.
viscous layer of carbohydrates that are covalently bound to the cell wall in
Gram-positive bacteria or often to lipids of the outer membrane in many
DOI:10.1111/1574-6976.12056
Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of
Editor:MiguelCamara high molecular weight polysaccharides contributing to virulence of many
human pathogens in the gut, respiratory tree, urinary tract, and other host tis-
Keywords sues, by hiding cell surface components that might otherwise elicit host
capsularpolysaccharides;glycosaminoglycans; immune response. This review highlights capsular polysaccharides that are
S
polysialicacid;bacterialpathogens;immune structurally identical or similar to polysaccharides found in mammalian tissues,
W
systemevasion;combatingantibiotic
including polysialic acid and glycosaminoglycan capsules hyaluronan, heparo-
resistance.
E san, and chondroitin. Such nonimmunogenic coatings render pathogens insen-
I sitive to certain immune responses, effectively increasing residence time in host
V
tissues and enabling pathologically relevant population densities to be reached.
E
Biosynthetic pathways and capsular involvement in immune system evasion are
R
described, providing a basis for potential therapies aimed at supplementing or

Y replacing antibiotic treatment.
G
O through covalently linked lipids that are inserted into
Introduction
the lipid bilayer of the membrane. This provides a sur-
L
O Bacterial capsular polysaccharides (CPSs) are major viru- face layer of water-saturated, high molecular weight
lence factors that confer protective effects to their bear- polysaccharides that limit desiccation in the face of
I
B ers against a wide range of environmental pressures, harsh environmental conditions, block infection by most
O most notably against the immune system during infec- bacteriophages, and thwart phagocytosis and other host
tion of their animal hosts. Although capsules are often immune responses by physically restricting access to cell
R
associated with descriptions of pathogenic bacteria due surface antigens. These polysaccharide cloaks are likely
C
to the large proportion of encapsulated invasive patho- rational targets for wide-spectrum therapeutic com-
I
M gens, nonpathogenic and commensal bacteria also benefit pounds aimed at replacing or supplementing antibiotic
from the ability to envelope themselves with a capsule treatment of microbial infections, as removal of the
(Hafez et al., 2009; Dasgupta & Kasper, 2010). In Gram- capsule exposes bacteria to routine immune clearance
negative bacteria, capsular polysaccharides are often pathways mediated frequently by activation of the
attached to the outer membrane at their reducing end complement system.
ª2013FederationofEuropeanMicrobiologicalSocieties. FEMSMicrobiolRev38(2014)660–697
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Masqueradingmicrobialpathogens 661
1999). Serotyping systems for other species were
Historical perspective
developed in a similar manner, but the relative ease of
The molecular compositions of CPSs vary extensively Gram-negative CPS structural characterization and the
between organisms and even between strains within a sin- genetic tractability of E. coli enabled more rapid develop-
gle species, but, despite this diversity, some species from ment of the E. coli antigen classification scheme. Owing
distinct orders have been shown to biosynthesize identical to their antigenicity in mammals, most CPS structures
CPS structures (DeAngelis, 1999; DeAngelis & White, elicit T lymphocyte-independent immune responses that
2002). The existence of highly homologous biosynthetic induce IgM antibody production but fail to stimulate
machinery for production of identical polysaccharides T-cell-dependent IgM-IgG switching, an important attri-
between microorganisms suggests that capsular gene clus- bute to ensure long-lasting immunity (Weintraub, 2003;
ters have been acquired through horizontal gene transfer; Avci & Kasper, 2010). However, purified CPSs from some
conversely, nonhomologous glycosyltransferases biosyn- of the most commonly isolated strains were determined
thesize identical polysaccharides in disparate organisms to be nonimmunogenic due to structural identity with
(Vann et al., 1981; Finne et al., 1983; Korhonen et al., human glycans (Edwards et al., 1982; Johnson, 1991;
1985; Jann & Jann, 1998), an occurrence that has likely H!erias et al., 1997). Capsule-deficient mutants of these
developed through functional convergent evolution strains generally exhibit decreased virulence, persistence,
(DeAngelis, 2002a, b) facilitated by interkingdom coevo- and serum sensitivity (Pluschke et al., 1983; H!erias et al.,
lution of prokaryotic pathogens with their eukaryotic 1997). As discussed in greater detail later, antibody gener-
hosts (Gagneux & Varki, 1999; Chen & Varki, 2010). ation proved difficult against purified mammal-like bacte-
Capsule structure diversity was originally investigated as rial CPSs composed of hyaluronan (HA), heparosan, or
part of a broad effort to classify bacterial strains based certain congeners of unsulfated chondroitin or polysialic
upon their interaction with human serum; that is, bacte- acid (PSA).
ria were serotyped through differentiation of their cell It should be noted here that there are reports of anti-
surface antigens (Lancefield, 1933). Serotyping is critical bodies generated against these CPSs under unique cir-
for understanding pathogenicity from medical, diagnostic, cumstances (Frosch et al., 1985; Jennings et al., 1985;
and immunologic perspectives and has remained the Kabat et al., 1986; Kro€ncke et al., 1990; Finke et al.,
dominant method for classifying strains of capsular bacte- 1991; Troy, 1992; Born et al., 1996). However, careful
ria. While studies in the early twentieth century demon- consideration should be given to possible antigenic
strated that polysaccharides present in the capsules of determinants for antibodies generated in such experi-
both Gram-negative and Gram-positive bacteria were ments and whether access to the epitopes would result
antigenically distinct from cellular protein fractions (Hei- in protective responses in vivo. If serum-accessible por-
delberger & Avery, 1923), research throughout the follow- tions of these CPSs are identical to mammalian glycans,
ing decades further differentiated and classified these it seems unlikely that antibodies could be elicited against
polysaccharidic antigens and ultimately implicated the these ‘self’ epitopes. In some cases, antibodies were
eponymous CPSs in elevated serum resistance and inhibi- raised in autoimmune animal hosts, where self-protec-
tion of granulocytic phagocytosis (Peterson et al., 1978; tion capacity was diminished due to immune dysregula-
Horwitz & Silverstein, 1980). tion (Bitter-Suermann et al., 1986). Immune response by
Complementary serological and clinical studies during healthy animal hosts requires other possible explanations
the latter half of the twentieth century identified a subset to clarify this paradox:
of streptococcal, staphylococcal, meningococcal, and Esc- 1 The CPS possesses an exposed antigenic determinant
herichia coli CPSs associated with increased virulence and not found in the corresponding mammalian glycan, such
widespread incidence in severe bacterial infections, pro- as a deacetylated amino sugar or terminal unsaturated
voking investigation of the relationship between CPS bond generated by a lyase (a class of enzymes acting to
structure and immunogenicity (Robbins et al., 1974; Kaij- cleave acidic polysaccharides through an eliminase mecha-
ser et al., 1977). Elucidation of the chemical structures of nism, in contrast to hydrolyzing glycosidases, Linhardt
various K-antigens (an alternative name for the CPS of et al., 1986) or some other nonself-chemical decoration.
E. coli) and apparent demarcation based upon their phys- 2 CPS purification exposes a nonmammalian antigenic
ical properties prompted development of a K-antigen determinant, like an anchoring moiety composed of a
classification system (Orskov et al., 1977) that was revised phospholipid or a monosaccharide or oligosaccharide lin-
over time to incorporate genetic and biomolecular evi- ker constituent not biosynthesized in mammals.
dence (Jann & Jann, 1997), ultimately being supplanted 3 The antigenic determinant spans a self and nonself-
by a more robust grouping scheme based on genetic, bio- domain on the purified CPS, thereby cross-reacting with
chemical, and molecular criteria (Whitfield & Roberts, the self-domain.
FEMSMicrobiolRev38(2014)660–697 ª2013FederationofEuropeanMicrobiologicalSocieties.
PublishedbyJohnWiley&SonsLtd.Allrightsreserved
662 B.F.Cressetal.
4 The antibody is not specific for the CPS but is cross-
Bacterial glycans
reacting due to structural similarity to the true antigen.
In one example, a human IgM class antibody reactive Bacteria produce an array of carbohydrates that are not
against PSA was shown to be nonspecific due to the limited to CPSs, and an understanding of these bacterial
antibody’s reactivity with polynucleotides, a scenario glycans is critical for appreciating the role of the CPS in
where cross-reactivity was enabled possibly due to simi- the pathogenesis of infection. As depicted in Fig. 1,
lar surface charge distributions in these two negatively Gram-negative bacteria possess an external layer of long
charged biopolymers (Kabat et al., 1986). An IgG anti- CPS chains that are covalently anchored by phospholipids
body against PSA was also isolated in autoimmune to the outer leaflet of the outer membrane, an asymmet-
NZB mice (Frosch et al., 1985). Nevertheless, evidence ric lipid bilayer with an external layer composed primarily
supports the conclusion that nonimmunogenic bacterial of lipid A (also known as endotoxin) and an internal
CPSs sharing structural identity with host glycans confer phospholipid layer. Anchored to the outer membrane by
an additional protective advantage over immunogenic, lipid A, the lipopolysaccharide (LPS) serves as a hydro-
nonhost CPSs. philic barrier to natural hydrophobic antibiotics and is
Due to their poor immunogenicity, mammal-like CPSs composed of three regions: (1) lipid A, a highly conserved
were originally considered nontypeable by traditional region that possesses two phosphorylated, b-1,6 linked N-
means. However, ‘typing’ of some nonimmunogenic CPSs acetyl-D-glucosamine (GlcNAc) saccharide residues bear-
was later achieved by screening strains against phages ing variable numbers and lengths of fatty acyl chains; (2)
only capable of attachment and subsequent infection the core oligosaccharide, which can be subdivided into
when a specific CPS is displayed on the bacterial surface, the inner and outer cores. The inner core is covalently
such as E. coli K1- and K5-specific phages (Roberts et al., bound to lipid A and possesses a species-dependent or
1986; Scholl et al., 2001). With increasing genetic charac- strain-dependent nonlinear oligosaccharide composed of
terization of CPS-producing strains, polymerase chain 3-deoxy-D-mannooctulosonic acids (KDO), heptoses, and
reaction and sequencing-based methods such as multilo- some nonglycan components such as pyrophosphoetha-
cus sequence typing (MLST) can now be used for fast nolamine (PPEtn). The outer core is linked to the termi-
and accurate molecular typing (Townsend et al., 2001; nal heptose of the inner core and possesses a more
O’Hanlon et al., 2004; Durso et al., 2005; Kong et al., variable nonlinear structure of primarily hexose residues;
2005; Zhu et al., 2012). Despite the utility of (3) the last region of the LPS is known in E. coli as
antibody-based serotyping, confounding variables at the the O-antigen due to its distinct antigenicity from the
immunologic level make molecular diagnosis an attractive K-antigen. The O-antigen is a repetitive glycan that varies
alternative. in composition and length between species and strains,
Capsular
polysaccharide
Lipopolysaccharide
Outer membrane
Periplasm
Inner membrane
Fig.1. Schematic cross-sectional representation of layers constituting the bacterial cell wall of a typical Gram-negative bacterium. The thick
external CPS layer conceals the bacterium to prevent desiccation, bacteriophage infection, complement-mediated killing, and
opsonophagocytosis. The black and white inset (top left) shows a quick-freeze, deep-etch scanning electron micrograph of the Gram-negative
organism Bacteroides thetaiotaomicron (Martens etal., 2009); this SEM image was originally published in The Journal of Biological Chemistry.
Martens EC, Roth R, Heuser JE & Gordon JI. Cover image. J Biol Chem. 2009; 284(27):cover. © the American Society for Biochemistry and
MolecularBiology.
ª2013FederationofEuropeanMicrobiologicalSocieties. FEMSMicrobiolRev38(2014)660–697
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Masqueradingmicrobialpathogens 663
but it is typically masked from the environment by the glycopolymers known as exopolysaccharides, which have
K-antigen. Finally, an oligopeptide-cross-linked lattice of long been considered determinants of biofilm physico-
alternating b-1,4 linked GlcNAc and N-acetylmuramic chemical properties (Costerton et al., 1987). Surprisingly,
acid (MurNAc) residues known as peptidoglycan is con- over the last decade, exopolysaccharides from certain
strained within the periplasmic space between the outer microorganisms have also been shown to inhibit biofilm
and inner (cytoplasmic) membranes. In many species, formation by other microbial species (Valle et al., 2006;
peptidoglycan sugar moieties are further modified after Kim et al., 2009; Nithya et al., 2010; Bendaoud et al.,
installation (Vollmer, 2008). Water-soluble b-glucan poly- 2011; Jiang et al., 2011). As an example of the diversity of
mers known as membrane-derived oligosaccharides bacterial glycans, the glycocalyx of a strain of E. coli can
(MDOs) are found near the inner membrane and are simultaneously possess six exopolysaccharides. In addition
decorated with negatively charged ethanolamine, phos- to biosynthesis of covalently bound O-antigen and K-
phoglycerol, and succinyl groups. These highly charged antigen, a linear heteropolysaccharide known as entero-
MDOs protect the inner cell membrane from low osmotic bacterial common antigen (ECA) is produced by all
conditions (Esko et al., 2009). Thus, glycans are critical members of the Enterobacteriaceae family and is also fre-
components of the cell wall that contribute to structural quently bound to the outer membrane. ECA consists of a
integrity and interaction with the environment (Com- conserved [?3)-a-Fuc4NAc-(1?4)-b-ManNAcA-(1?4)-
stock & Kasper, 2006; Fig. 2). As Gram-positive bacteria a-GlcNAc-(1?]n repeating trisaccharide unit that can be
do not possess an outer cell membrane, the peptidoglycan bound by the reducing end of GlcNAc to the LPS core,
layer is thicker compared with Gram-negative bacteria. anchored to the outer membrane by a phosphate bridge
Lipoteichoic acids anchor the inner layers of peptidogly- with diacylglycerophosphate, or secreted in a water-solu-
can to a glycolipid extending from cytoplasmic mem- ble, cyclized, and partially 6-O-acetylated (on GlcNAc)
brane, while wall-associated teichoic acids tether form with polymerization degree typically between 3 and
additional outer layers together through covalent linkages 6 trisaccharide repeats (Erbel et al., 2003; Fregolino et al.,
to MurNAc residues. CPSs biosynthesized by Gram-posi- 2012). Other exopolysaccharides, such as colanic acid
tive bacteria can be anchored to the inner membrane, to (known as M-antigen), are also secreted into the environ-
oligopeptide cross-linkers within peptidoglycan, or to ment by many E. coli strains. Although only some colanic
GlcNAc residues in the peptidoglycan lattice (Hanson & acid (CA) remains loosely associated around the cell,
Neely, 2012). especially when constrained within a biofilm during sub-
In addition to LPS and CPS, many species of Gram- optimal growth, an E. coli strain has been isolated in
negative and Gram-positive bacteria also biosynthesize which CA is ligated to the outer core of the LPS in place
and secrete an assortment of high molecular weight of the O-antigen (Meredith et al., 2007). Another
(a) (b)
O-Antigen
repeat unit
Outer core
Glycocalyx
Inner core
(c)
Fig.2. Glycan-centricschematicoftypical
Gram-negativecellwallcomponents. K-Antigen
Outer membrane
repeat unit
Membraneproteinsandothercellwall
constituentsareneglectedforsimplicity.
Peptidoglycan
(a)Cellwallcross-section.(b) Poly-KDO
Membrane derived linker
Lipopolysaccharide.(c)Capsular oligosaccharides
polysaccharide.Abbreviationsareasfollows:
Inner membrane
Lyso-PG=lyso-phosphatidylglycerol,
GlcNAC=N-acetylglucosamine,
MurNAc=N-acetylmuramicacid,
Lipid A GlcNAc Heptose Glucose Glucose substituted
GalNAc=N-acetylgalactosamine,
with ethanolamine,
KDO=3-deoxy-D-mannooctulosonicacid, Phospholipid MurNAc KDO GlcA phosphoglycerol, or
PPEtn=pyrophosphoethanolamine, Lyso-PG GalNAc PPEtn Peptide crosslinker succinylgroup
GlcA=glucuronicacid.
FEMSMicrobiolRev38(2014)660–697 ª2013FederationofEuropeanMicrobiologicalSocieties.
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664 B.F.Cressetal.
exopolysaccharide of E. coli, commonly secreted by many and N. meningitidis, while HA is found in the capsules of
other bacteria as well, is the adhesin poly-b-1,6-GlcNAc, strep throat and necrotizing fasciitis-causing S. pyogenes
a homopolymer that encourages adherence and biofilm and P. multocida, the etiological agent of fowl cholera
formation (Wang et al., 2004, 2005). Finally, E. coli and and many other mammalian and bird diseases. Bacteria
other bacteria also biosynthesize and secrete an exopoly- containing these CPSs are compiled in Table 1 and are
saccharide known as bacterial cellulose, or poly-b-1,4-glu- included in the discussion where relevant.
can, as a component of the bacterial extracellular matrix
(Zogaj et al., 2001). It can be inferred that dynamic regu-
Molecular mimicry and coevolution
lation of this suite of bacterial extracellular glycans allows
sampling of many glycocalyx states to adapt to a wide In evaluating the ability of host-like CPSs to evade the
range of environments (Meredith et al., 2007). immune system, it is important to understand which host
While Gram-positive CPS structures are diverse and tissues contain similar molecules and how pathogens
can be difficult to characterize, Gram-negative CPS struc- coevolved with humans to enable such mimicry. The first
tures are comparatively simple and have thus been more capsule type addressed in this review is known as polysia-
amenable to categorization (Whitfield, 2006). In spite of lic acid or PSA, consisting of N-acetylneuraminic acid
the depth and breadth of chemical and serological analy- (Neu5Ac) monomers joined with various glycosidic link-
sis of CPS structures, however, there remains a dearth of ages. PSA capsules produced by strains of K1 E. coli,
evidence regarding linkage of Gram-negative CPS to the N. meningitidis serotype B, Moraxella nonliquefaciens, and
cell. A very recent and fascinating report has resolved this Mannheimia haemolytica (previously Pasteurella haemoly-
longstanding enigma for a class of model E. coli and Nei- tica) A2 are characterized by a-2,8 glycosidic linkages
sseria meningitidis strains, demonstrating that capsules (Adlam et al., 1987; Devi et al., 1991), while PSA pro-
assembled through a common ATP-binding cassette duced by other microorganisms possess a-2,9 glycosidic
(ABC) transporter pathway are biosynthesized on a nearly linkages (N. meningitidis serotype C strains) or alternating
conserved lyso-phosphatidylglycerol anchoring moiety a-2,8 and a-2,9 glycosidic linkages (K92 E. coli strains;
through an oligo-KDO linker, presumably guiding the Glode et al., 1977; Lifely et al., 1986). In mammals, PSA
translocation of such CPSs to the cell surface in a CPS- is an a-2,8-linked polysaccharide on neural cell adhesion
and organism-independent manner (Willis et al., 2013). molecule (NCAM), which is found on the surface of neu-
CPSs in this category are known as Group 2 K-antigens rons, glial cells, and natural killer cells, a type of lympho-
in E. coli, but all characterized N. meningitidis strains cyte functioning as an integral part of the innate or
possess a homologous transport system described below. nonspecific immune response (Rutishauser, 2008; Chang
Despite the variety of bacterial glycans, CPSs comprised et al., 2009). Increased surface polysialylation leads to
of nonimmunogenic polysaccharides are of primary medi- charge repulsion and is associated with decreased NCAM
cal interest due to their conspicuous ability to evade the adhesion in animals (Rutishauser et al., 1985) and resis-
immune system. Those sharing identity with human poly- tance to phagocytosis by PSA capsular bacteria (King
saccharides have been cataloged in a number of patho- et al., 2007). Structural identity of mammalian PSA with
genic species, but the most well-characterized CPS E. coli K1 and N. meningitidis type B CPSs – particularly
structures are found in E. coli, N. meningitidis, Pasteurella embryonic NCAMs, which contain more than 50 a-2,8
multocida, and Streptococcus pyogenes. It is interesting to Neu5Ac repeating units, compared with a much lower
compare P. multocida, Avibacterium paragallinarum, and degree of polysialylation in adult NCAMs (Jann & Jann,
E. coli as strains of all species have been found to produce 1998) – contributes to the neuroinvasiveness of these
chondroitin and heparosan, while certain strains of incredibly virulent neuropathogens (Robbins et al., 1974;
P. multocida also possess HA capsules. Although Sarff et al., 1975). The PSA capsule is thought to enable
P. multocida and A. paragallinarum are predominantly traversal of the blood–brain barrier (Kim et al., 2003),
animal pathogens rather than human pathogens, the abil- thus leading to high rates of morbidity and mortality in
ity of these microorganisms to produce identical nonim- neonatal meningitis and serious neurological conditions
munogenic capsules to E. coli through similar yet distinct in survivors of the disease (Kaper et al., 2004).
genetic and enzymatic processes warrants inclusion of the The other three host-like CPSs discussed in this review
species in this review. Moreover, the diseases caused by are considered glycosaminoglycans (GAGs), or negatively
these organisms in livestock pose economic threats and charged, linear polysaccharides identical to the backbones
cause concern regarding the contribution of antibiotic- of GAGs found in animals and composed of a repeating
laden livestock feed to the spread of antibiotic resistance. core disaccharide unit, comprised of an uronic acid resi-
Identical PSA capsules are also produced between differ- due linked to an amino sugar (Ho€o€k et al., 1984).
ent species, including meningitis-causing strains of E. coli Although the monomeric sugar precursors constituting
ª2013FederationofEuropeanMicrobiologicalSocieties. FEMSMicrobiolRev38(2014)660–697
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Masqueradingmicrobialpathogens 665
Table1. PathogenicbacteriapossessingnonimmunogenicCPSsthatareidenticaltohumanandanimalglycans
Serotype/
CPS GAG Organism* capsuletype Disease(s)(organism) Reference(s)
Polysialicacid No Escherichiacoli K1 Meningitis,urinarytractinfection, Silveretal.(1988)
diarrhea,septicemia(human)
Polysialicacid No Neisseriameningitidis B Meningitis(human) Finneetal.(1983)
Polysialicacid No Moraxellanonliquefaciens Endopthlamitis,sepsis,meningitis, Bøvreetal.(1983),Devietal.(1991)
endocarditis(human) andRafiqetal.(2011)
Polysialicacid No Mannheimia A2 Bovinerespiratorydisease(bovine) Adlametal.(1987)and
(formerlyPasteurella) Riceetal.(2007)
haemolytica
Chondroitin† Yes Escherichiacoli K4 Urinarytractinfection, Orskovetal.(1985),Moxley&
diarrhea(human);diarrhea(bovine) Francis(1986)and
Rodriguezetal.(1988)
Chondroitin Yes Pasteurellamultocida TypeF Fowlcholera(avian) Rimler&Rhoades(1987)
Chondroitin Yes Avibacteriumparagallinarum GenotypeI Coryza(avian) Wuetal.(2010)and
Zhaoetal.(2010)
Heparosan Yes Escherichiacoli K5 Urinarytractinfection(human) Minshewetal.(1978)and
Zingleretal.(1990)
Heparosan Yes Pasteurellamultocida TypeD Pneumonia(porcine) Ewersetal.(2006)
Heparosan Yes Avibacteriumparagallinarum GenotypeII Coryza(avian) Wuetal.(2010)
Hyaluronan Yes Streptococcuspyogenes Scarletfever,pharyngitis(human) Wesselsetal.(1991)andRalph&
Carapetis(2013)
Hyaluronan Yes Streptococcusequi Septicemia,meningitis,endocarditis Wibawanetal.(1999)and
ssp.zooepidemicus andarthritis(bovine,porcine, Weietal.(2012)
ovine,andcanine)
Hyaluronan Yes Streptococcusdysgalactiae Streptococcaltoxicshock Calvinhoetal.(1998)and
ssp.equisimilis syndrome(human) Hashikawaetal.(2004)
Hyaluronan Yes Streptococcusuberis Mastitis(bovine) Almeida&Oliver(1993)and
Almeidaetal.(2013)
Hyaluronan Yes Streptococcusequissp.equi Upperrespiratorytract Anzaietal.(1999)
infection(equine)
Hyaluronan Yes Pasteurellamultocida TypeA Respiratorydisease(bovine,feline) Borrathybayetal.(2003b)
andEwersetal.(2006)
Hyaluronan Yes Avibacteriumparagallinarum Coryza(avian) Sawata&Kume(1983)and
Byarugabaetal.(2007)
*Inrecentyears,therehasbeenmuchconfusionregardingdelineationofStreptococcusspeciesandsubspeciesduetoimprecise,muddled,and
archaic classification systems (Jensen & Kilian, 2012). Similar scenarios occurred with Avibacterium and Mannheimia. Organism names are pro-
videdasoriginallyreportedunlessaclearindicationofmisclassificationwasdetected.
†Fructosylated.
these core disaccharide units are conserved in GAGs, the potential (Kamhi et al., 2013). Symbolic representations
disaccharide units in the animal GAGs heparan sulfate of bacterial CPS structures and related animal glycan
and chondroitin sulfate are not strictly repeating because structures are illustrated in Fig. 3.
they are variably sulfated and acetylated within a single In particular, the CPSs produced by K4 E. coli and
chain. The dominant disaccharide unit in the polysaccha- P. multocida type F strains are related to CS, a class of
ride and the distribution of specific disaccharide types, sulfated GAG characterized by a [?4) b-D-glucuronic
glycosidic linkage configurations, molecular weight, acid (GlcA) (1?3) N-acetyl-b-D-galactosamine (GalNAc)
degree of sulfation and acetylation, and in some cases (1?] disaccharide repeat, where position and extent of
n
degree of epimerization define the class of GAG and con- sulfation are tissue and organism dependent (Rodriguez
tribute to heterogeneity within each class. GAGs exhibit et al., 1988; Volpi, 2007; Volpi et al., 2008). While K4
their numerous biologic activities by interacting with pro- CPS GlcA residues are substituted with bisecting b-fruc-
teins including growth factors, chemokines, and adhesion tofuranose units between C2 of fructose and C3 of GlcA,
molecules (Capila & Linhardt, 2002; Linhardt & Toida, the fructose is acid labile, and K4 CPS is thought to exist
2004). The interactions between GAGs and pathogens can as an unsubstituted backbone in certain low pH environ-
also represent the first line of contact between pathogen ments (Jann & Jann, 1990). Conversely, P. multocida type
and host cell and are crucial to a pathogen’s invasive F CPS is identical to the unsulfated chondroitin precursor
FEMSMicrobiolRev38(2014)660–697 ª2013FederationofEuropeanMicrobiologicalSocieties.
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666 B.F.Cressetal.
Chondroitin Chondroitin sulfate* Heparan sulfate/Heparin†
β4 β3 β4 β3 β4 β3
NRE RE Heparosan α4 β4 α4 β4 α4 β4 α4 α4
β4 β3 β4 β3 β4 β3 NRE RE
HOOC HO OH NRE RE NS NS NS3S 2S NS
O O O O 4S/ 0S/ 4S/ 0S/ 4S/ 0S/ 4S/ 6S 6S 6S
NRHEFOruβc4tosββy33lOaHtβe4dO βc3hoβn4drβoβ33iNtHinARcE OHHO6OSOC26SS/ OO6SR2 O26SSO/R46SOR6O26SS/NHA6cSO NORHEOOCα4 βO4 αO4 β4 α4OHβO4 RE OHHOOOC OOR2OR3O ONRHO6Y O
HO OOHOOOC OOH HOO OHONHAcO HOOOC OOH O OR4 OOR6 O HO OH NHAc O HOOOC RO2OOH RO3O NHOOYR6 O
R2O NHAc
OHOH
Polysialic acid Polysialic acid Hyaluronan Hyaluronan
α8 α8 α8 α8 α8 α8 α8 α8 α8 α8 α8 α8 β4β3 β4β3 β4β3 β4 β3 β4 β3 β4β3
NRE RE NRE RE
Bacterial NRE RE NRE RE
HOOC HOOC HOOC HOOC capsular Human
HOOAcHN OHOOH HOOAcHN OHOOH O HOOAcHN OHOOH HOOAcHN OHOOH O polysaccharide glycan OHOHOOC OOHHOO ONHHOAc O OHOHOOC OOHHOO ONHHOAc O
GalNAc GlcNAc Neu5Ac GlcA IdoA Fructose Acid-labile glycosidic linkage
Fig.3. Symbolic representations and chemical structures of glycans described in this review. Nonimmunogenic bacterial CPSs and structurally
related animal glycans exhibited side-by-side to demonstrate similarity between backbones. In the case of chondroitin sulfate (CS) and heparan
sulfate/heparin,bacterialCPSstructuresareidenticaltoprecursorsofthematurehumanglycansdepictedhere.Ofnote,arelatedGAGknown
asdermatansulfatealsosharestheunsulfatedchondroitinbackboneasabiosyntheticprecursor,but,unlikeCS,someglucuronicacidresiduesin
the chain are epimerized to iduronic acid. CS type B possesses iduronic acid residues, so it is sometimes classified as dermatan sulfate.
Conversely,HAandPSAstructuresareidenticalinmicrobialCPSandmaturehumanglycans.*R2,4,6=HorSO3!;†R2,3,6=HorSO3!,Y=SO3!
orAc(Ac=COCH ).Detaileddisaccharidestructureshavebeenreportedelsewhere(Sugahara&Mikami,2007;Changetal.,2012b).
3
of animal CS (DeAngelis et al., 2002). Considering the disaccharide units, is identical to the mammalian precur-
limited patterns of O-sulfo group substitution within the sor for heparin and heparan sulfate (Ly et al., 2011;
disaccharide-repeating unit of animal CS (Sugahara & Fig. 3). Heparan sulfate is typically found on the cell
Mikami, 2007), it is apparent that the order of the sulfo- membrane and in the extracellular matrix as a component
nation reactions is important in CS biosynthesis and that of proteoglycans (Gallagher, 1989). Heparin, a highly sul-
O-sulfo groups in certain positions can preclude the fated variant of heparan sulfate, is biosynthesized as an
activity of downstream sulfotransferases (Schiraldi et al., intracellular proteoglycan, serglycin (Li et al., 2012). The
2010). CS occurs in animals as an O-linked glycan chain, GAG heparin is a widely used anticoagulant pharmaceuti-
covalently bound to serine residues of proteins, through a cal (Capila & Linhardt, 2002). Although the natural func-
specific tetrasaccharide linkage, resulting in glycoconju- tion of heparin is not well understood, its release from
gates known as proteoglycans (Esko, 1999). This class of the granules of mast cells is localized to damaged tissue
GAG is found primarily in the extracellular matrix where and contributes to wound healing and defense against
one or more CS chain is attached to an array of core pro- opportunistic infection (Zehnder & Galli, 1999). The
teins affording proteoglycans with various structural and nontemplate-driven biosynthesis of heparan sulfate and
regulatory roles. Proteoglycans mediate a myriad of physi- heparin affords a diverse range of disaccharide units from
ological interactions such as cellular recognition, commu- a modest number of biosynthetic enzymes (Fig. 3).
nication, migration, adhesion, and proliferation (Thelin The GAG known as hyaluronan or HA is structurally
et al., 2013). identical in animals and in capsules of S. pyogenes groups
The CPS produced by K5 E. coli strains such as Bi A and C, P. multocida type A, and some other species of
8773-41 and the probiotic strain Nissle 1917 (Lodinov!a- bacteria (DeAngelis, 1999). HA GAG consists of an
Z#aadnikov!a et al., 1992) and also by P. multocida type D unmodified [?4) b-D-GlcA (1?3) b-D-GlcNAc (1?]n
strains is composed of heparosan (Vann et al., 1981; disaccharide-repeating unit in both animals and bacteria.
DeAngelis & White, 2002). Heparosan, comprised of [? In animals, this high molecular weight polysaccharide is
4) b-D-GlcA (1?4) a-D-GlcNAc (1?]n repeating the predominant GAG in the extracellular matrix and is
ª2013FederationofEuropeanMicrobiologicalSocieties. FEMSMicrobiolRev38(2014)660–697
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Masqueradingmicrobialpathogens 667
found in high quantities in skin, connective tissues, carti- the negatively charged surface of phagocytes, thus
lage, synovial fluid, and the vitreous humor of the eye increasing the unfavorable interaction when phagocytosis
(Ho€o€k et al., 1984; Dougherty & van de Rijn, 1992). or complement-mediated lysis occurs (Moxon & Kroll,
The role of these host-like, or ‘self’, capsules enveloping 1990; Kuberan & Linhardt, 2000). According to van Oss
the surfaces of invasive pathogens seems quite clear: and Gillman, the phagocytic cells such as polymorpho-
molecular mimicry enables such pathogens to evade an nuclear leukocytes (PMNs), monocytes, and macrophag-
immune system that has learned to ignore self molecules es repel the encapsulated bacteria due to the net Lewis
based on cell surface interactions. As glycans are ubiqui- AB repulsion between the hydrophilic outer layers (Kla-
tous on cell surfaces (Gallagher, 1989), they are likely the iner & Geis, 1975; van Oss et al., 1975), which reduce
first molecules contacted by pathogens that utilize cellular the surface tension between the phagocytic cell and the
adhesion during infection, so pathogens bearing these bacterium (Moxon & Kroll, 1990). For example, the cell
capsules have an evolutionary advantage. The question surface of Staphylococcus aureus became less hydrophilic
that remains is how humans and other animals have after removing the capsule and its phagocytic uptake
evolved to combat these camouflaged pathogens and how was enhanced (van Oss et al., 1975). A similar phenom-
pathogens continually evolve to successfully colonize their enon was observed with the encapsulated strain of
animal hosts. In a series of papers, Ajit Varki argues that Salmonella typhimurium, which resists phagocytosis, but
genetic evidence suggests pathogens – which evolve orders when unencapsulated, it is readily phagocytized (Cunn-
of magnitude faster than their hosts due to horizontal ingham et al., 1975). Noneffective contact can often lead
gene transfer, high mutation rates, fast growth, and short to the failure of phagocytic engulfment. More intuitive
life spans – develop self-CPSs through convergent evolu- is simple charge–charge repulsion between the negative
tion, where the glycosyltransferase genes responsible for charge of the CPS and the glycocalyx of the phagocytic
biosynthesizing the glycans in pathogen and host are not cell. The more highly charged the CPS, the more
homologous in most cases (Gagneux & Varki, 1999). likely a bacterium is to avoid opsonophagocytosis
Considering that glycosyltransferases are highly conserved (Moxon & Kroll, 1990). Poor phagocytosis of a ‘smooth
within the host and yet biosynthesize highly diverse gly- surface’ may directly result from the physical surface
can structures and distributions in different cell types and properties instead of biologic interaction of capsules
tissues, coupled with the combinatorial style of glycan with phagocytic signaling and complement-mediated
interactions and the ability to maintain functional speci- molecules. Direct experimental testing of this hypothesis
ficity when a participating glycan is modified, Varki also remains challenging.
argues that sexual reproduction-enabled mutations in The interaction between the CPS of the bacterial sur-
host glycosyltransferases and subsequent change in glycan face and the host’s complement system is also a key
profile allow these multicellular organisms to adapt to contributor to bacterial virulence. In the early stage of
pathogenic pressure. Furthermore, Varki speculates that the immune response, the control and defense mecha-
the coevolution of pathogens and their hosts has not only nism of the host are contingent on the classic and alter-
tailored the diversity of glycan structures and expression native complement pathways. The classic pathway is
patterns, but that pathogenic pressures stemming from usually initiated by antigen–antibody binding. The C1
host-like capsules contribute significantly to speciation of complement complex, which is a multimolecular prote-
multicellular organisms (Varki, 2006). ase consisting of three subunits C1q, C1r, and C1s, trig-
gers the classical pathway of complement, first binding
to the aggregated antibody molecule, then sequentially
Immune response and clearance of
cleaving and activating the complement protein C4 and
encapsulated pathogens
proenzyeme C2 to form a C3 convertase, C2bC4b (Jann
Both evasion of complement-mediated killing and failure & Jann, 1997). This process is regulated by C4-binding
of being ingested by phagocytic cells enhance the viru- protein C4bp (Roberts, 1996) and is usually retarded
lence of the CPS. The polysaccharide capsules are effec- during the encapsulated bacterial invasion. The C3 con-
tive physical barriers that protect the bacteria from vertase then converts C3 to the activated C3b, which
being killed. The fact that bacteria capsules are com- will be deposited on the bacterial cell surface. This pro-
monly hydrophilic and negatively charged diminishes cess is controlled by factors B and H of the alternative
their removal through phagocytosis. The hydrophilic pathway (Jann & Jann, 1997). The alternative pathway
nature causes high-level hydration and reduces the can be activated in the absence of antibody binding to
surface tension at the phagocyte and bacterium interface the bacterial surface and therefore is very important in
(Kuberan & Linhardt, 2000). Additionally, the negatively immunity to encapsulated or unencapsulated bacteria.
charged polysaccharides on the bacterial surface repel In other words, the alternative pathway provides a way
FEMSMicrobiolRev38(2014)660–697 ª2013FederationofEuropeanMicrobiologicalSocieties.
PublishedbyJohnWiley&SonsLtd.Allrightsreserved
668 B.F.Cressetal.
for the immune system to kill bacteria in the blood in fructosyl linkage under low pH environment transforms
the absence of specific antibodies. The alternative K4 CPS into nonimmunogenic chondroitin (Jann & Jann,
pathway utilizes the serum protein C3b, which is then 1997).
activated by serum factor B, D, and properdin (Moxon
& Kroll, 1990), to form convertase C3bBb that amplifies
Capsular polysaccharide transport,
the complement cascade for more C3 conversions and
genetics, biosynthesis, and role in
C3b deposition (Roberts, 1996). The activation of C3b
immune system evasion
results in a ligand targeting specific receptors on PMNs
or macrophages. The binding of a C3b opsonized The chemical properties and immunogenicity of CPSs are
microorganism to the complement receptor on PMNs dictated by variations in number, order, and diversity of
or macrophages initiates phagocytosis and ultimately monosaccharide constituents, anomeric centers (a- or b-),
killing of the encapsulated bacteria. In addition, follow- glycosidic linkage positions, absolute configuration (L or
ing C3b deposition, the sequential activations of C5 to D), ring forms (pyranose or furanose), degree of chemical
C9 form a membrane attack complex that directly leads modification (O-acetylation, for example), and overall
to the lysis and death of some Gram-negative bacteria conformation (Mazmanian & Kasper, 2006). There is a
(Moxon & Kroll, 1990; Roberts, 1996). wide range of capsule types among bacterial orders and
This bacterial defense mechanism and the subsequent even within a single species. For instance, strains belong-
response of complement-mediated bacterial killing by the ing to one of the most well-studied CPS-producing spe-
host can be blocked at numerous sites by CPSs avoiding cies, E. coli, are known to biosynthesize c. 80 CPS
serum-mediated killing and enhancing virulence. Some structures. The number of known capsule types increases
capsules protect the bacteria from being attacked by steric dramatically when considering other genera, but capsules
mechanisms. Bacteria such as pneumococci promote C3b in other organisms are less well characterized due to lim-
deposition on the bacterial cell surface underneath the ited biochemical studies and relative genetic recalcitrance.
capsule, shielding it from recognition by the phagocytic Nevertheless, studies in the model capsular species E. coli
cell (Winkelstein, 1981). Some bacterial capsules interrupt suggest that the capsule assembly pathways are compara-
the binding of C3b to the bacterial surface by affecting tivelylimitedinscope,whereadiverseassortmentofCPSs
regulatory proteins, such as factor B and H (Loos, 1985; is assembled and translocated to the cell surface using
Cross, 1990). Capsules that exert such a defense mecha- identical strategies. Biochemical and genetic evidence in
nism usually contain N-acetylneuraminic acid (Neu5Ac) Gram-negative bacteria paints a picture of a veritable
because it contains a factor H binding site. The stimula- orchestra of catalytic enzymes, structural proteins, and
tion of H-C3b correspondingly decreases the amplifica- transport proteins interacting in a transmembrane com-
tion convertase C3bBb, leading to failure of the plex that spatially and temporally organizes biosynthesis
complement cascade (Moxon & Kroll, 1990). Some cap- andtransport.Themodularityofthecooperatingsubcom-
sules cannot bind to factor B, thus causing more H-C3b plexes allows distinct CPS biosynthetic enzymes, com-
formation (Winkelstein, 1981). Strains such as E. coli K1, plexed at the inner membrane, to utilize identical
E. coli K92, N. meningitides types B and C, and Group B transport systems for translocation of disparate CPS.
Streptococcus polysaccharides have capsules that inhibit Whitfield and coworkers recently showed an ABC-trans-
alternative complement activation by these mechanisms porter-dependent pathway common to some E. coli and
(Stevens et al., 1978; Wessels et al., 1989). N. meningitidis strains results in the biosynthesis of
The mimicry of the CPS structure to substances within unique CPSson a common anchor structure (Williset al.,
the host serves as a virulence factor preventing bacteria 2013). This apparently ensures successful CPS transport
phagocytosis. A CPS can mimic a similar structure found and outer-membrane attachment. Similarly, another com-
within the host representing ‘self’, and therefore, both monly conserved transport system, known as the Wzy-
avoid recognition as foreign and circumvent triggering dependent pathway, shares the ability to assemble CPSs
the host immune response (Kuberan & Linhardt, 2000). withrelaxedspecificityforCPSstructure.Althoughawide
The CPS K1 has the same poly-a-2,8-Neu5Ac (PSA) range of bacteria utilize the ATP-dependent and Wzy-
structure as carbohydrate portion of NCAM, required for dependent pathways for CPS assembly, the majority of
organogenesis and neural cell growth (Finne, 1982; Kub- experimental evidence has been acquired in E. coli.
eran & Linhardt, 2000). Similarly, the CPS K5 strain of Homologous genes between species have been identified
E. coli shares the same structure as mammalian heparo- by sequence similarity in many cases rather than by func-
san (Navia et al., 1983). An X-ray diffraction study tional characterization. Hence, this section of the review
showed that the K4 capsule was poorly immunogenic willfocusonE. coliasa modelsystemanddrawcompari-
due to its similar helix structure to CS. The removal of sonsbetweenrelatedbacteriawhererelevant.
ª2013FederationofEuropeanMicrobiologicalSocieties. FEMSMicrobiolRev38(2014)660–697
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Masqueradingmicrobialpathogens 669
do not share identity with animal glycans, they elicit an
Transport pathways
immune response and are thus out of the scope of this
In E. coli, CPS structures have been classified into four review. The reader is directed to two excellent reviews
groups. Group 1 and 4 CPS structures (as well as colanic compiling recent research in this area (Whitfield, 2006;
acid) are found in enteropathogenic (EPEC), enterotoxi- Reid & Cuthbertson, 2012).
genic (ETEC), and enterohemorrhagic E. coli (EHEC) Group 2 and 3 E. coli CPSs are produced in strains
strains and are assembled through what is known as the commonly associated with extraintestinal infections
Wzy-dependent pathway. This pathway is distinct from (ExPEC), while all known E. coli CPS structures sharing
the so-called ABC-transporter-dependent pathway that is identity with animal glycans belong to Group 2. It is also
responsible for assembly and transport of Group 2 and 3 interesting to note that Group 2 and 3 E. coli CPSs share
CPSs and that is described in detail later. While uronic certain similar structure and assembly characteristics with
acid sugars are common to Group 1 CPS repeat units, strains of N. meningitidis, P. multocida, Haemophilus
Group 4 CPS repeats are characterized by the presence of influenzae, and Campylobacter jejuni (Whitfield, 2006). In
acetamido sugars. Despite this apparent structural distinc- contrast to Group 1 and 4 CPSs, the repeat units of
tion between Group 1 and 4 CPSs, both are polymerized Group 2 and 3 CPSs exhibit extensive variation in struc-
and transported to the cell surface in a similar manner. ture. Similar to the Wzy-dependent transport system, the
In the Wzy-dependent system, serotype-specific repeating ABC-transporter dependent system expressed by Group 2
units are assembled from cytosolic sugar precursors and and 3 E. coli strains has relaxed specificity for CPS struc-
linked to undecaprenyl diphosphate by glycosyltransferas- ture, successfully transporting very distinct structures
es, unique to the specific type of CPS being synthesized, across the cell wall. A striking difference compared with
which are embedded in the cytoplasmic membrane. Indi- Wzy-dependent assembly is that Group 2 and 3 CPSs,
vidual undecaprenyl diphosphate-linked repeating units assembled by ABC-transporter-dependent pathways, are
are then transferred across the inner membrane to the completely polymerized in the cytoplasm and then trans-
periplasm by a flippase, Wzx, which also passes the repeat ported across the cell wall to the outside of the cell. CPSs
unit to an integral membrane protein known as Wzy. of this class are elongated by processive, CPS-specific gly-
Wzy processively catalyzes addition of these individual cosyltransferases that are colocalized to the cytoplasmic
Group 1 and 4 CPS repeat units to the reducing end of surface of the inner membrane with other proteins
the growing polysaccharide chain, which elongates in the belonging to the coordinated biosynthetic-transport com-
periplasm without being released by Wzy until chain ter- plex. Details regarding polymerization initiation are not
mination (Yi et al., 2006). Wza, Wzb, and Wzc are fully resolved, but CPSs from N. meningitidis group B,
responsible for control of chain length and export from E. coli K1, and E. coli K5 strains (all Group 2 type cap-
the periplasmic face of the inner membrane to the cell sules) were recently shown (Willis et al., 2013) to be
surface. In Group 4 strains, longer polysaccharide chains linked to a well-conserved lyso-phosphatidylglycerol
can be incorporated into the LPS structure and effectively (lyso-PG) terminus by a poly-b-KDO linker. It should be
anchored by lipid A, although these K-antigens are classi- noted that slight variation in fatty acyl chain length and
fied as K to distinguish their unique attachment mech- number of KDO repeats was measured within single cul-
LPS
anism (Whitfield, 2006). In Group 1 strains, shorter tures and between organisms. For instance, the single
polysaccharide chains can also form K , but longer fatty acyl chain of lyso-PG in most cultures varied
LPS
chains are known to assemble capsules without covalent between saturated C16 (palmitoyl-PG) or monounsatu-
attachment to LPS. Although the outer-membrane pro- rated C18 (oleoyl-PG), but one culture produced diacyl-
tein Wzi had been implicated in attachment of Group 1 PG with either two C16 chains (dipalmitoyl-PG), two
CPSs (specifically the K30 antigen) to the outer mem- C18 chains (dioleoyl-PG), or one C16 adjacent to a C18
brane (Rahn et al., 2003), the exact mechanism was chain (palmitoyl-oleoyl-PG). Furthermore, the number of
unknown until recently. A paradigm shift in understand- KDO monomers exhibited interstrain and intrastrain vari-
ing CPS attachment resulted from a study that concluded ation between 5 and 9 KDO repeats. This discovery sug-
K30 CPS remained associated with the outer surface of gests that the common glycolipid carrier is the anchor by
the cell due to interactions with an outer-membrane lec- which the ABC-transporter guides Group 2 CPSs from
tin, Wzi, that captures secreted CPS and serves as a the cytoplasm to the outer membrane. However, the
nucleation site for further CPS recruitment (Bushell et al., mechanism by which the glycolipid carrier is assembled
2013). Wzy-dependent capsules are also biosynthesized in and attached to the nascent polysaccharide remains unde-
Klebsiella pneumoniae, and much of the molecular insight termined. Comparatively little is known about assembly
for early steps in this pathway came from studies of Sal- and transport of Group 3 CPS, but high sequence homol-
monella enterica O-antigen assembly. As CPSs in this class ogy with Group 2 transport machinery suggests that the
FEMSMicrobiolRev38(2014)660–697 ª2013FederationofEuropeanMicrobiologicalSocieties.
PublishedbyJohnWiley&SonsLtd.Allrightsreserved